The long-term objectives are the evaluation and redesign of adoptive immunotherapy (Al) protocols based on the fundamentals of lymphocyte trafficking. The original plan was refocused on the most novel and promising Aim (Aim 2) in light of the 3-year award period. Initial work revealed that tumor-reactive Ti pre-effector cells were concentrated in tumor draining lymph node (TDLN) cells synthesizing bindings sites (ligands) for the vascular adhesion receptors P- and E-selectin. Selectin-ligand synthesis occurred on a small subset of the activated T-cells implying that selective culture of these cells (Plighigh cells) might improve anti-tumor activity in vivo. A para-magnetic fractionation procedure, using recombinant human 1gM: murine P-selectin chimera (lgMPsetlHM), was devised to test the hypothesis. The large amounts of IgMPselHM needed for processing 5-10 x 108 cells/experiment fueled development of a high-capacity transient-transfection system. Selective culture of the Plighigh cells increased suppression of pulmonary micrometastases (3-day) by 10-20 fold and greater than 70 fold for MCA-205 sarcomas and B16 melanomas, respectively. 50 percent suppression of MCA-sarcoma metastases required 0.1-0.3 x 106 cultured Plighigh, 2-4 x 106 cultured unfractionated and greater than 9 x 106 cultured Pliglow TDLN per animal. 50 percent suppression of B16-melanoma mets required 1 x 106 cultured Plighhigh versus 70 x 106 cultured unfractionated TDLN per animal. Thus, TDLN fractionation using adhesion receptors involved in Ti trafficking markedly enhances the therapeutic benefit from Al. This renewal builds on these promising results with the following Aims. (1) Identify the PIighigh subsets in TDLN that produce anti-tumor activity during culture. The Plighigh cells isolated from TDLN are heterogeneous. Therefore, this aim determines which cell types (a/b T-cells, NK, NK-T, 13-cells, g/d T-cells APCs) influence anti-tumor activities. (2) Identify the effector mechanisms that increase the potency of cultured Plighigh TDLN. The contributions of T-cell subsets, vascular adhesion receptors and chemokines will be investigated for responding metastases in various locations. (3) Determine whether treatment with pro-inflammatory cytokines improves the recruitment and anti-tumor activities of cultured Plighigh cells. In other words, can one increase the delivery and clinical benefit of cultured Plighigh cells by inducing an inflammatory response in tumor-bearing organs or individual macrometastases? (4) Evaluate selectin-based sorting in humans using specimens from patients receiving adoptive immunotherapy for disseminated renal cell carcinoma.